1. http://www.setcor.org/conferences/NANOTECH-DUBAI-2013
Nanotech Dubai 2013
International Conference
Proceeding

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Nanotech Dubai 2013 International Conference and Exhibition
Synthesis of doxorubicin containing drug carriers and their in-vitro performance on HeLa and
HCT116 cells
Christian Schmidt1
, Nurdan Doğangüzel1,2
, Stefanie Klöpzig1
, Sophia Rehfeldt1
, Sascha Behne1
,
Raphael-Jose da Silva1
, Jens-Peter Krause2
, Mont Kumpugdee-Vollrath2
, Joachim Storsberg*1
1
Fraunhofer Institute Applied Polymer Research (IAP), Geiselbergstrasse 69, 14476 Potsdam-
Golm, Germany; 2
Department of Pharmaceutical Engineering, Beuth University of Applied
Sciences Berlin, 13353 Berlin, Germany.
* Corresponding author: phone: +49331-568-1321; facsimile: +49-331 568-33-1321; email:
joachim.storsberg@iap.fraunhofer.de.
Abstract
To further the options available for a more efficient anticancer therapy, we modified lipid carriers
of the anticancer agent doxorubicin (Dox). In utilizing a previously reported differential sensitivity
of HeLa and HCT116 cells to 1µM Dox, our results show a more than 50% decreased survival
rate of the more resistant HeLa cells to 1µM Dox, delivered via our new carrier system; the blank
carrier system does not impair the survival rate of the more sensitive HCT116 cells. This
internally validated assay may be regarded as advantageous in determining the efficacy of drug
delivery systems.
Keywords: drug carrier, efficacy, evaluation, cell culture, internal validation
Introduction
The delay-adjusted cancer death rate in the United States of America decreased more than 1%
for both males and females in the time frame encompassing the years 2005 through 2009.
Factors contributing to this decline include access to health care and advances in detection
and/or treatment options for malignant lesions [1].
Leaving the issue of early-stage detection of tumors aside, delivery of the needed dosage of
anticancer medication is a goal yet to be reached. Currently, much higher doses of drugs need
to be administered to patients, leading to inconveniences caused by side effects. For example,
patients have benefited from the anticancer activity of doxorubicin (Dox) for more than 30 years,
although its inherent cardio-toxicity limits the dosages administered [2-4]. Concordantly,
research efforts are directed at the development of systems to deliver drugs more efficiently to
the target [5, 6]. One of these new formulations is a liposomal Dox formulation for intravenous
injection purposes, which gained FDA approval [7, 8]. Caring physicians are, however, forced to
reduce administered dosages of liposomal Dox due to profound to severe manifestations of the
Palmar-Plantar Erythrodysesthesia [9, 10]. Examples of alternative nanoparticular-based Dox
delivery systems already undergoing Phase III trials illustrate not only commercially viable

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avenues but, more importantly, efforts to fully address the unmet clinical need of new drug
delivery systems [11, 12].
How different dosages of Dox affect cancer cells is not fully understood. Complexity is added by
observations where acute lymphoblastic leukemia cells (Molt-4) showed signs of apoptosis
concomitant with RNA synthesis but no apparent sign of oxidative stress after twelve hours of
exposure to the pharmacologically relevant concentration of 1µM Dox. Higher doses of Dox (>
3µM) led to a significyntly reduced apoptosis and administration of >100µM Dox resulted in
inhibition of RNA polymerase and increased oxidative damage in the model system tested [13].
As our understanding of the molecular events leading to the development of cancerous lesions
improves, more and more published evidence emerges in support of a so far not contested
notion of an involvement of members of the p53 family in the many steps leading to the genesis
of cancers, including but not limited to an aberrant function of p53 itself. As for p53, anomalous
function of this tumor suppressor may be manifested in a wide range, including a functionally
mutated form of p53 or even a complete loss of the p53 gene [14-17]. For example, degradation
of p53 is targeted by the Human Papillomavirus (HPV) E6 oncogene in the case of invasive
cervical cancers [18-21]. A direct consequence would be a reduced transcriptional activity of a
p53 reporter plasmid in cells derived from such invasive cervical carcinoma. Using HeLa cells
with their survival tied to the expression of E6, it was found that the p53 reporter plasmid appears
to be more responsive to Dox treatment of the HPV negative HCT-116 cells in comparison to the
HPV18 positive HeLa cells [22-26]. One logical extension of the above-discussed published
evidence could be that HeLa cells are less susceptible to the effects of pharmacologically
relevant doses of Dox (1µM) than HCT-116 cells.
To test this hypothesis, we treated HeLa and HCT-116 cells with Dox solubilized in growth
medium or delivered via our new carrier system. Our results confirm a reduced sensitivity of
HPV E6 positive HeLa to treatment with 1µM Dox in comparison to the HPV-negative HCT-116
cells. Furthermore, our results show that drug loaded nanoparticular carrier systems decrease
the survival of HeLa cells by more than 50%, while unloaded nanoparticles do not impair the
survival rate of the more sensitive HCT-116 cell line. In conclusion, our data support the notion
of using an internally validated tissue culture system to test the efficacy of new anticancer drugs
and/or delivery systems.
Body
1. Methods
We obtained all tissue culture plastic, from TPP, Trasadingen, Switzerland. The following sterile
preparations were obtained from Biochrom AG, Berlin, Germany: DMEM [27], PBS without
Mg2+
/Ca2+
[28], RPMI-1640 [29], FBS, ultrapure water, 100x stock solutions of sodium pyruvate,
nonessential amino acids and Penicillin/Streptomycin and 10x stock solution of Trypsin/EDTA.
HeLa and HCT116 cells (gifted by Dr. Norbert Nass, Otto-von-Guericke-University, Institute of
Pathology, Magdeburg, Germany) were maintained in RPMI-1640 containing 10% heat-
inactivated FBS [30], 4mM L-glutamin, 2mM sodium pyruvate and 500 U Penicillin and 500 U
Streptomycin. Tissue culture, cryopreservation and thawing of cells were performed according to
[31].

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The following chemicals were used for the preparation of lipid-based carrier systems [32,33]: 2,6-
di-tert-butyl-4-methylphenol (BHT, ACROS, Lot # A0203142), Soybean oil (Roth, Karlsruhe,
Germany, Lot # 352187856), Cetyl palmitate (Roth; Lot # 452162569), Sodium tetradecyl sulfate
(Alrdich, Munich, Germany, Lot # MKBK1230V), Miglyol 812 (Caesar & Loretz, Hilden, Germany,
Lot # 12078605), Polysorbate 80 (Roth, Lot # 292187273 and 302187273), Lecitin (Roth, Lot #
312189810) and Doxorubicin (Molecula, Munich, Germany, Lot # 201249 and Sigma, Munich,
Germany, Lot # SLBC8638V). We examined mean hydrodynamic particle size and zeta
potential using samples diluted 1:2,500 in particle-free water as described [34]. For the in-vitro
performance test of synthesized carrier systems on HeLa cells, we seeded 104
HeLa cells per
cm2
culture area on day -3 into a well of a 12-well plate and grew them for 3 days (until day 0) in
1ml of growth medium. On day 0, growth medium was replenished and supplemented with
either Dox from a 10mg/ml stock solution in growth medium, or either loaded carrier system to
deliver the amounts of Dox indicated in the figures or comparable amounts of unloaded carriers.
Viable and dead cells were determined after incubation for 3 days (day 3) as described above.
For tests involving HCT116 cells, we seeded 2.5x103
HCT116 cells per cm2
of culture area on
day -2 into a well of a 12-well plate and grew them for 2 days (until day 0). On day 0, growth
medium was replenished and supplemented as described above. Viable and dead cells were
determined after incubation for 2 days (day 2) as described above. The relative survival rate of
the cells is plotted in reference to untreated cells (set to 100%). All experiments were performed
in triplicates with mean values and
standard deviations plotted using
Microsoft Excel®.
2. Synthesis and characterization
of carrier systems
For carrier preparation, we followed
[32] with modifications: The aqueous
phase consisted of 15.2g water
(supplemented to 0.1% Dox final
concentration in the case of Dox
loaded carriers), 2g polysorbate 80,
whereas the lipid phase consisted of
300mg lecitin, 500mg BHT, 3.75g
Cetyl palmitate and either 3.75g
soybean oil or Migylol 812. Both
phases were preheated in individual
containers to 70°C under constant
agitation on an IKA magnetic stirrer
at 600 rpm. Over a time period of 15 min, we then added dropwise the aqueous phase to the
lipid phase at a constant temperature of 70°C under constant agitation at 600 rpm, followed by 5
min of further agitation at 70°C. As preparation for the sonication step, we removed the heat
source and stirred the mixture until room temperature (25°C) was reached. The mixture was
sonicated 5 times for 5min each with a 10sec break between individual sonication steps (40%
Figure 1: Mean hydrodynamic particle size distribution of freshly
prepared delivery systems.

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amplitude on a Sonoplus Mini 20 from Brandt Electronics). Final adjustments of the pH to 8 ± 0.1
were performed with 1M NaOH followed by successive passages of the product through 1µm
and 0.45µm membrane filters. STS was added to the preparation at a molar ratio of 1mol Dox to
1.25 mol STS followed by incubation for 3 days at 4°C in the dark and subsequent sonication for
3min (40% amplitude on a Sonoplus Mini 20 from Brandt Electronics), according to [33].
At first, we assayed for mean hydrodynamic particle size distribution [34] directly after completion
of particle synthesis (Figure 1). In agreement with [33], the mean hydrodynamic particle size of
the materials after synthesis was determined to be around 200 nm and of unimodal distribution
(Figure 1). Another determination of the mean hydrodynamic particle size was performed to
address one of the major limitations of drug delivery systems created by emulsion techniques,
the susceptibility of the particles to Ostwald ripening [35, 36]. As shown in Figure 2, no profound
changes in the mean hydrodynamic particle size of the particles was detected after 7 months of
storage at 4°C in the dark. This virtually unchanged mean hydrodynamic particle size distribution
of the filtration sterilized formulation of drug carriers lends support to the recommendation of [33]
to use filtration through a 0.22µm membrane filter as sterilization method for this particular
formulation of drug carriers. By extension, this result ties in nicely with the notion that
sterilization methods of drug carrier formulations should be determined on a case-by-case
manner [33].
Figure 2: Mean hydrodynamic particle size distribution of delivery systems after 7 months of storage at 4°C in the dark.

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The zeta potential of Dox loaded soybean-based carriers changed to -37.92 ± 0.86 mV from -
52.36 ± 0.34 mV for unloaded particles. As for miglyol-based carriers, Dox-loading altered the
zeta potential from -35.54 ± 0.26 mV from the blank reference to -27.98 ± 0.92 mV. These
results suggest that loading of either carrier system with Dox resulted in a decreased negative
charge with a calculated respective absolute difference of 14.44 mV for the soybean-based
formulation and 7.96 mV for miglyol-based carriers. This decrease in negativity in response to
Dox loading of carrier systems is also in agreement with observations of different carrier systems
[35], although differences in the absolute numbers may be attributed to the properties of the
individual system. Taken together, our results are in agreement with published evidence and
support the notion that the synthesized carrier systems were successfully loaded with Dox.
To further describe the carrier formulations obtained, we assayed for the release of Dox by
means of dialysis assays [36]. In brief, we placed 5ml of the indicated drug carriers (equal
amounts of Dox loaded) in a dialysis bag (Mw cut off: 4 to 6Da, thickness: 28µm and with a
nominal filter rate of 3.5, obtained
from Roth) and dialyzed this against a
constant volume of 25ml buffer (pH
7.4) consisting of 10 vol-% ethanol,
15 vol-% polysorbate 80 and 70 vol-
% Sorensen buffer under constant
agitation at 600 rpm at 37°C [37, 38].
At time points 1, 2, 4, 7, 24, 32 and
48h, 2ml of the medium was drawn to
determine the amount of Dox by
comparing the optical density of a reference to the sample. Plotting [Dox] in µM against time in
hours resulted in the graph shown in Figure 3. As shown in Figure 3, both preparations
displayed a sigmoidal drug release profile as assayed via a dialysis assay. This ties in well with
published evidence of an observed sigmoidal release of a model compound, phenyl
isothiocyanate, from chitosan-encapsulated solid lipid nanoparticles made of stearic acid,
Tween-80 and sodium taurocholate [39]. Our observations indicate that the Dox release from
miglyol-based carrier systems reached the first plateau of the release profile at approximately 7h
compared to 4h in the case of soybean oil-based formulations. Both formulations showed a
sigmoid Dox release between 24 and 32 h. Notably, an initial burst of released drug is seen in
the release profile of both drug carrier systems. In agreement with published literature [40], our
interpretation of this observation is that the unrestricted diffusion of the drug through the drug
delivery system into the surrounding medium is the rate-limiting step.
3. In vitro performance of synthesized carrier systems on HeLa and HCT116 cells
Effects caused by recognition of and reaction with intravenously administered carrier systems by
the phagocytic system may result in undesirable responses of a patient to treatment approaches
[41]. Using solid lipid nanoparticles and primary mouse peritoneal macrophages, Schöler et al.
[42] found that medium concentrations up to 0.1% of carrier formulations based on soybean oil,
lecithin and tween 80 and with particle sizes ranging from around 200nm to 5µm did not reduce
the viability of primary murine peritoneal macrophages after 20h incubation as assayed by MTT
Figure 3: Release of Dox as a function of time in a dialysis assay.

7. Page 6 of 10
tests. In support of [25], HeLa cells did not respond to 1µM Dox with a reduced survival rate in
comparison to 0.1µM Dox throughout our assays, whereas HCT116 cells display a roughly 5 fold
decreased survival in the presence of 1µM Dox in comparison to 0.1µM Dox (Figures 4 and 5).
To account for the cytotoxic properties of empty carrier systems, we added comparable amounts
of empty carriers as were needed to the delivery of the concentration of Dox in the indicated
treatments with loaded nanoparticles. As shown in Figure 4 for soybean oil-based carrier
systems, delivery of 0.1µM Dox increased the killing efficiency 3 fold in the case of HeLa cells
and nearly 5 fold for HCT116 cells in comparison to free Dox in growth medium. Miglyol-based
drug carriers, however, proved to be less effective in the killing of HeLa (decrease by 2 fold at
1µM Dox) and HCT116 cells (decrease by 2 fold at 0.1µM Dox) in comparison to free Dox in
growth medium). In sum, the results depicted in Figure 4 suggest that soybean oil-based and
Dox loaded carrier systems display a higher efficacy than miglyol-based drug delivery systems.
Figure 4: In vitro performance of soybean oil or miglyol-based carrier systems on HeLa and HCT116 cells.

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As shown in Figure 5 for STS/soybean oil-based carrier systems, delivery of 0.1µM Dox
increased the killing efficiency 5 fold in the case of HeLa and HCT116 cells in comparison to free
Dox in growth medium. Miglyol-based drug carriers, however, proved to be less effective in the
killing of HeLa (decrease by 2 fold at 1µM Dox) and HCT116 cells (decrease by 5 fold at 0.1µM
Dox) in comparison to free Dox in growth medium). In sum, these results (Figures 4 and 5)
suggest that the killing efficacy of HeLa cells by soybean oil-based carrier systems increased
when STS is present in the carrier formulation. This, however, cannot be observed in the case of
miglyol-based formulations.
Conclusion
Here we show the synthesis and characterization of sovbean oil or miglyol-based drug carrier
systems with an unchanged particle size of around 200nm regardless of loading with doxorubicin
and/or storage for 7 months at 4°C in the dark. Addition of STS at a molar ratio of 1mol Dox to
1.25 mol STS increased the killing efficacy of the doxorubicin-resistant cell line HeLa as well as a
Figure 5: In vitro performance of STS containing soybean oil or miglyol-based carrier systems on HeLa and
HCT116 cells.

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doxorubicin-sesitive cell line (HCT116) using soybean oil-based carrier systems but not the
miglyol-based drug delivery systems. These observations present an advantage over existing
literature where a nontoxic drug carrier system is reported to overcome the doxorubicin
resistance in MDA-MB 231 cells but appears to be less efficient in killing a doxorubicin-sensitive
cell line (MCF-7) [43].
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